ABSTRACT One of the tasks of the kidney is to maintain cellular and total body acid-base homeostasis by reclaiming filtered bicarbonate and making and saving new bicarbonate. The electrogenic sodium/bicarbonate cotransporter (NBCe1) plays a significant role in reabsorbing bicarbonate in the proximal tubules, where more than 80% of filtered bicarbonates are reclaimed. Electrogenic function of NBCe1 is essential for this bicarbonate reabsorption, producing a driving force for sodium and bicarbonate exit across the basolateral membranes of the proximal tubule cells. Altered activities of NBCe cause severe proximal renal tubule acidosis as well as glaucoma and cataracts. The long-term goal of this project is to elucidate molecular mechanisms for electrogenic sodium/bicarbonate transport of NBCe1. We propose to perform an integrated structure/function analysis of NBCe1 and identify the structural domains and amino acid residues that are essential for electrogenicity. The experimental designs are i) to systematically construct a series of chimeric transporters from NBCe1 and the electroneutral sodium/bicarbonate transporter NBCn1 that moves sodium and bicarbonate into the cell, and ii) to construct point mutants of NBCe1 by sequence comparison with NBCn1. There are three specific aims. In Aim 1, we will identify the transmembrane domains of NBCe1 that affect electrogenicity. Chimeric transporters will be constructed by swapping individual transmembrane domains of NBCe1 with the homologous regions of NBCn1. The function of those chimeric transporters will be analyzed by measuring the pH recovery rate and bicarbonate-dependent currents in Xenopus oocytes expressing the proteins. In Aim 2, we will determine amino acid residues affecting electrogenicity. We will select the residues, within identified domains, that might severely alter electrogenic function and mutate them. In Aim 3, we will distinguish functionally essential transmembrane domains and amino acids from structurally supportive ones. This will be done by reconstituting identified domains/amino acids of NBCe1 into the electroneutral transporter. The proposed work will help develop molecular models of electrogenic sodium/bicarbonate movement via NBCe1